title: Advances in multiplexed MRM-based protein biomarker quantitation toward clinical utility.
abstract: Accurate and rapid protein quantitation is essential for screening biomarkers for disease stratification and monitoring, and to validate the hundreds of putative markers in human biofluids, including blood plasma. An analytical method that utilizes stable isotope-labeled standard (SIS) peptides and selected/multiple reaction monitoring-mass spectrometry (SRM/MRM-MS) has emerged as a promising technique for determining protein concentrations. This targeted approach has analytical merit, but its true potential (in terms of sensitivity and multiplexing) has yet to be realized. Described herein is a method that extends the multiplexing ability of the MRM method to enable the quantitation 142 high-to-moderate abundance proteins (from 31mg/mL to 44ng/mL) in undepleted and non-enriched human plasma in a single run. The proteins have been reported to be associated to a wide variety of non-communicable diseases (NCDs), from cardiovascular disease (CVD) to diabetes. The concentrations of these proteins in human plasma are inferred from interference-free peptides functioning as molecular surrogates (2 peptides per protein, on average). A revised data analysis strategy, involving the linear regression equation of normal control plasma, has been instituted to enable the facile application to patient samples, as demonstrated in separate nutrigenomics and CVD studies. The exceptional robustness of the LC/MS platform and the quantitative method, as well as its high throughput, makes the assay suitable for application to patient samples for the verification of a condensed or complete protein panel. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Percy
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Chambers
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada.
authors.forename: Juncong
authors.initials: J
authors.lastname: Yang
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada.
authors.forename: Darryl B
authors.initials: DB
authors.lastname: Hardie
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada; Department of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada. Electronic address: christoph@proteincentre.com.
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D015415:Biomarkers / Q000032:analysis*; D001798:Blood Proteins / Q000378:metabolism*; D002318:Cardiovascular Diseases / Q000175:diagnosis* / Q000378:metabolism; D002853:Chromatography, Liquid; D015331:Cohort Studies; D005260:Female; D006801:Humans; D007553:Isotope Labeling; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D054647:Nutrigenomics; D010446:Peptide Fragments / Q000032:analysis*; D034941:Upper Extremity; D055815:Young Adult
chemical_list: D015415:Biomarkers; D001798:Blood Proteins; D010446:Peptide Fragments
keywords: MRM; Multiple reaction monitoring; Multiplexed; Plasma; Protein quantitation; Undepleted